Calcium Dependent Protein Kinases (CDPKs) are important calcium regulated signal transducing proteins in plants. These proteins contain a protein kinase domain, which is regulated through interactions with autoinhibitory and calmodulin-like domains. Autophosphorylation has been observed in numerous CDPKs from various sources, but its mechanistic consequences and physiological importance have not been well characterized. Preliminary results, showing that an autophosphorylation site in the calmodulin like domain is conserved in three CDPKs from different species, support the physiological importance of this phenomenon. We propose to determine the extent, location, kinetics, and physiological significance of autophosphorylation of two diverse CDPK family members, CPK4 and CPK28, from Arabidopsis thaliana. To accomplish these goals, CPK4 and CPK28 protein will be expressed and purified to homogeneity using bacterial expression systems. Protein produced in this way will be used to find the site(s) of autophosphorylation, and to evaluate autokinase kinetics and stoichiometry. Protein will also be provided to our collaborators for X-ray crystallography. The autophosphorylation sites will be altered by site directed mutagenesis, and the effects of these changes will be evaluated in vitro and in vivo.